A new diagnostic microarray (Check-KPC ESBL) for detection and. identification of extended-spectrum beta-lactamases in highly resistant

Transcription

1 JCM Accepts, published online ahead of print on 8 June 2011 J. Clin. Microbiol. doi: /jcm Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved A new diagnostic microarray (Check-KPC ESBL) for detection and identification of extended-spectrum beta-lactamases in highly resistant Enterobacteriaceae Ina Willemsen 1, Ilse Overdevest 2, Nashwan al Naiemi 3, Martine Rijnsburger 3, Paul Savelkoul 3, Christina Vandenbroucke-Grauls 3, Jan Kluytmans 1,3 on behalf of the TRIANGLe study group Department of Medical Microbiology and Infection control, Amphia hospital, Breda, The Netherlands 2 Department of Infection Control, Sint Elisabeth Hospital, Tilburg, The Netherlands 3 Department of Medical Microbiology and Infection control, VU University Medical Center, Amsterdam, The Netherlands Running title: ESBL detection using a microarray Word count: abstract 50 words, text words Footnote 1: Checkpoints BV, Wageningen supplied the materials for the microarray There was no external funding for this project Footnote 2: None of the authors has a potential conflict of interest 1

2 Corresponding author: Jan Kluytmans, Consultant Microbiologist Laboratory of Microbiology and Infection Control Amphia hospital PO Box 90158; 4800 RK BREDA; The Netherlands TEL: / FAX: The TRIANGLe study group: Department of Infection Control, Academic Medical Center Amsterdam,E. Lommerse andl. Spanjaard; Laboratory for Microbiology and Infection Control, Antonius Hospital, Nieuwegein, B. Vlaminckx; Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen,A. Voss; Department of Infection Control, Catharina Hospital, Eindhoven, M. Wulf; Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, M. Vos;Laboratory for Microbiology and Infection Control, Roosendaal, the Netherlands, R. Wintermans;Laboratory for Microbiology and Infection Control, Bergen op Zoom, G. Andriesse; Department of Infection Control, Medical Center Leeuwarden, Leeuwarden, J. van Zeijl;Laboratory for Microbiology and Infection Control, Reinier de GraafGroep, Delft, the Netherlands, E. van der Vorm;Department of Infection Control, Sint Elisabeth Hospital, Tilburg, A. Buiting; Department of Medical Microbiology and Infectious Diseases, University Medical Center Nijmegen, Nijmegen, P. Sturm;Department of Medical Microbiology and Infectious Diseases, University Medical Center Utrecht, Utrecht, H. Blok anda. Troelstra. Abstract 2

3 The performance of a microarray for the detection of extended-spectrum betalactamases was determined on a collection of 638 highly resistant Enterobacteriaceae coming from 18 hospitals. The microarray had a significantly higher specificity than the phenotypic assays. It also detects carbapenemases and characterises the resistance genes, providing a better epidemiological insight Keywords: Extended-spectrum beta-lactamase, microarray, laboratory detection 3

4 The worldwide prevalence of extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) is increasing rapidly (1). The control of ESBL-E is difficult as the resistance genes are located on plasmids and may be transferred between species and even different genera of Enterobacteriaceae (2). The rapid laboratory detection of this resistance trait is important to guide antimicrobial therapy and to take appropriate infection control measures. We evaluateda ligation-mediated amplification in combination with a microarray to detect and characterize ESBL-Ein a contemporary collection of Enterobacteriaceae from a representative sample of hospitals in The Netherlands Multicenter prospective surveillance was performed in 18 Dutch hospitals during 6 months in All newly identified patients with highly resistant Enterobacteriaceae were included. The criteria for highly resistant Enterobacteriaceae are defined in the Dutch national guideline for the control of highly resistant microorganisms (3). Susceptibility tests were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (4). Presence of ESBL productionwas determined as previously described (5). All Escherichia coli, Klebsiellaspp., Proteus mirabilis, Salmonella spp. and Shigellaspp. (Group I) were tested with ceftazidime and Cefotaxime +/- clavulanic acid. All Enterobacterspp., Serratiaspp., Providenciaspp., Citrobacterfreundii, Morganellamorganii and Hafniaalvei (Group II) were tested with cefepime +/- clavulanic acid (6). When Etest results were non-determinable, a disk diffusion test (Roscodiagnostica, Taastrup, Denmark) was performed with the double disk methodology, using a similar algorithm. DNA isolation was performed with the Easymaq system (Biomerieux, Marcy l Etoile, France) according to manufacturer s instructions. The Check-KPC ESBL Array 4

5 (Checkpoints, Wageningen, the Netherlands) was used as previously described (6). bla SHV, bla TEM and bla CTX-M genes were amplified by PCR (7). Sequence analysis and alignments were performed with the Bionumerics 6.01 software (Applied Maths, Sint- Martens-Latem, Belgium) and online with BLAST program, and the information on All isolates with concordant phenotypic and genotypic ESBL detection results were considered identified correctly. For discordant findings, results of sequencing were considered the gold standard. 638 highly resistant Enterobacteriaceae were included (Table 1). Based on the phenotypic tests, 359 (55.9%) isolates were considered ESBL producers. The microarray detected one or more ESBL genes in 345 of the 638 (54.1%) isolates. Overall phenotypic and genotypic results for 590 out of 638 (92.5%) isolates were concordant. In group I, the majority (24/30) of discordant results had a positive phenotypic test and a negative micro-array result. In group II the majority (12/18) had a negative phenotypic test and a positive micro-array result. Table 2 shows the performance of the phenotypic and microarray tests when the results of sequencing are incorporated into the gold standard. For Group I, the sensitivities were comparable but the microarray was more specific. For group II, the microarray was more sensitive and the specificities were comparable. Seven of the false-positive phenotypic tests were negative upon retesting and six of the false-negatives were positive. Also both false-positive microarray results were negative upon retesting and five false-negative microarray results were positive on retesting. In the supplemental table all discordant findings are shown. The 345 isolates, which produced ESBL according to the microarray had various types of ESBL-genes (Table 3). More than half of the group I 5

6 isolatesenterobacteriaceae had bla CTX-M-1 family ESBL-genes, whereas in group II bla CTX-M-1 family were present it a minority. The most prevalent ESBL types in group II were bla CTX-M-9 family (61.6%). Some isolates contained more than one ESBL-gene. bla TEM co-existed once with a bla CTX-M-1 family ESBL gene and once with a bla CTX-M-9 family genes both in group I organisms. bla SHV was found six times in combination with bla CTX-M-1 family in group I organisms and nine times in combination with bla CTX- M-9 family in group I (n=7) and group II (n=2) organisms. One K. oxytocaisolate contained three different genes (bla TEM, bla SHV and bla CTX-M-9 family). In addition to ESBL genes, the microarray detects carbapenemase genes: these were detected in two K. pneumoniaeisolates, both from the same patient. The patientwho had been hospitalized in Greece after a traffic accident, was subsequently transferred to a general hospital in The Netherlands and from there to a University hospital. Both hospitals participated in our survey. Resistance to carbapenems had not been detected in the diagnostic laboratory with the standard laboratory procedures but was confirmed upon retesting. The Etest MIC for ertapenemwas 24.0 mg/ml This evaluation showed that the diagnostic microarray was more accurate than the current phenotypic methods to detect ESBL genes in a representative sample of clinical isolates. This microarray has recently been evaluated on three collections of selected isolates containing the majority of known ESBL and KPC genes (7,8,9). In the first evaluation (7), it detected 95% of the isolates that contained an ESBL gene and did not produce false positive results. The second evaluation (8) included a welldefined collection of ESBL and KPC producers and confirmed the ability of the array to detect most resistance genes. In one case the array failed to detect a KPC gene, 6

7 but the authors concluded that plasmid instability was the most likely explanation for this negative result. The third study tested the array on 106 Gram-negative strains(9). Thefollowing sensitivities and specificities were recorded:bla SHV, 98.8% and 100%; bla TEM, 100% and 96.4%; and bla CTX-M and bla KPC, 100% and 100% respectively. These promising results from the analytic evaluations were confirmed in our study. The false-positive phenotypic tests observed in Group I were often negative upon retesting. This reflects the subjectivity involved with the interpretation of the results. In Group II, chromosomal AmpC-production is a known pitfall for the phenotypic methods resulting in a substantially reduced specificity (10). As we did not include all known ESBL genes in the sequencing reactions there is a possibility that some of thefalse positive phenotypic tests are in fact true positives whichwere not detected by the molecular tests. However, the ESBL genes that were not included are rare.in conclusion, there were only few failures of the array to detect ESBL-genes and the specificity of the microarray was superior to the phenotypic tests, which makes this commercially available microarray a highly reliable tool to detect and identify ESBL genes in the clinical setting. In addition, it also detects the presence of carbapenemase genes, which are nowadays considered to be the most important threats of antimicrobial resistance (11). Finally, the array identifies the type of ESBL that is present, as shown in Table 3. The epidemiology of ESBL-E is rapidly changing and it is poorly understood how the resistance genes are spreading and which reservoirs are involved (12). This new tool will likely improve insight into the epidemiology of resistance genes, which may be an aid in the further control of resistance. 7

8 156 References ) Canton, R., A. Novais, A. Valverde, E. Machado, L. Peixe, F. Baquero, and T. M. Coque. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin.Microbiol.Infect. 2008;14Suppl 1: ) Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. ClinMicrobiol Rev. 2005;18: ) Kluytmans-VandenBergh M.F.Q, Kluytmans J.A.J.W, Vos A. Dutch guideline for preventing nosocomial transmission of highly resistant micro-organisms (HRMO). Infection. 2005;33: )Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: 15th information supplement. Wayne, PA: CLSI; 2005: M100-S15) ) Stuart JC, Leverstein M, al Naiemi N, members of the ESBL working party from the NVMM (2008) Guideline for screening and confirmation of Extended Spectrum Beta-Lactamases (ESBL) in Enterobacteriaceae. Available at accessed February 2010 Februari 18 th ) Sturenburg, E., I. Sobottka, D. Noor, R. Laufs, and D. Mack. Evaluation of a new cefepime-clavulanate ESBL Etest to detect extended-spectrum beta-lactamases in an Enterobacteriaceae strain collection.j.antimicrob.chemother. 2004;54:

9 ) Cohen Stuart J, Dierikx C, Al Naiemi N, et al. Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligationmediated amplification with microarray analysis. J AntimicrobChemother. 2010;65: ) Naas T, Cuzon G, Truong H, Bernabeu S, Nordmann P. Evaluation of a DNA microarray, the check-points ESBL/KPC array, for rapid detection of TEM, SHV, and CTX-M extended-spectrum beta-lactamases and KPC carbapenemases. Antimicrob Agents Chemother.2010;54: ) Endimiani A, Hujer AM, Hujer KM, et al. Evaluation of a commercial microarray system for detection of SHV-, TEM-, CTX-M-, and KPC-type beta-lactamase genes in Gram-negative isolates. J ClinMicrobiol. 2010;48: ) Potz NAC, Colman M, Warner M, Reynolds R and Livermore DM. False-positive extended-spectrum ß-lactamase tests for Klebsiella oxytoca strains hyperproducing K1 ß-lactamase. J AntimicrobChemother. 2004;53: ) Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis. 2009;9: ) Coque TM, Baquero F, Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill. 2008;13:

10 Table 1: Distribution of bacterial species Group I (n=501) Group II (n=137) Species Number (%) Species Number (%) Escherichia coli 332 (66.3) Enterobacter cloacae 87 (63.5) Klebsiella pneumoniae 93 (18.6) Citrobacter freundii 24 (17.5) Proteus mirabilis 41 (8.2) Morganella morganii 9 (6.6) Klebsiella oxytoca 29 (5.8) Serratia marcescens 6 (4.4) Salmonella spp. 3 (0.6) Citrobacter spp. 6 (4.4) Shigella spp. 2 (0.4) Enterobacter aerogenes 3 (2.2) Pantoea spp. 1 (0.2) Providencia spp. 2 (1.5)

11 Table 2: Results of the phenotypic tests and microarray testing after resolution of discordant results Gold standard ESBL negative ESBL positive Total Phenotypic: Group I ESBL negative ESBL positive Total Sensitivity 98.2%* Specificity 92.8% # Phenotypic: Group II ESBL negative ESBL positive Inconclusive Total Sensitivity 83.1%* Specificity 92.4% $ Microarray: Group I ESBL negative ESBL positive Total Sensitivity 97.2% Specificity 99.5% # Microarray: Group II ESBL negative ESBL positive Total Sensitivity 100% Specificity 98.5% $ * and # and $ : p <.05 11

12 Table 3: Identification of extended spectrum beta-lactamase genes with the microarray in Enterobacteriaceae of Group I and Group II. 216 Group I (%) Group II (%) Total 217 (%) 218 TEM 27 (9.5) 3 (3.8) 30 (8.2) 219 SHV 47 (16.5) 20 (25.3) (18.4) 221 CTX-M1 153 (53.7)* 6 (7.6)* 159 (43.7) 222 CTX-M2 0 (0) 4 (5.1) 4 (1.1) 223 CTX-M9 58 (20.4)* 45 (57.0)* (28.3) 225 CTX-M8/25 0 (0) 1 (1.3) 1 (0.3) 226 Total 285 (100) 79 (100) 364 (100) *: The difference between Group I and Group II is statistical significant for CTX-M1 and CTX-M9 12